Peptide binding by class II histocompatibility is a critical event in the generation of CD4+ T cells. Previous studies supported by this project demonstrated that the non-polymorphic MHC glycoprotein, HLA-DM catalyzes peptide loading and exchange reactions. The pH of endosomal compartments of antigen presenting cells also regulates peptide binding by controlling the conformation of class II molecules and regulating DM- catalyzed peptide exchange reactions. In this competing continuation application, experiments are proposed to further investigate factors that regulate peptide binding by MHC class II molecules with emphasis on characterizing the function of HLA-DM. The specific aims are to: 1) Characterize the role of membrane anchoring in HLA-DM catalytic activity; 2) Investigate the role of hydrogen bonds involving peptide main chain atoms in MHC class II structure, peptide binding, and DM activity; 3) Further characterize the role of pH and other factors in regulation of class II peptide loading; 4) Evaluate potential peptide-editing and chaperone functions of DM; 5) Analyze the role of CLIP and other domains of II in invariant chain functions. Overall, these experiments are expected to substantially advance our understanding of peptide binding by class II histocompatibility molecules and factors that regulate peptide binding under physiological conditions. A detailed understanding of this fundamental process is required for a complete picture of the elements that shape the general nature and specificity of the CD4+ T cell response.